Resistance alleles in soybean

ABSTRACT

The present invention relates to methods and compositions for identifying, selecting and/or producing a soybean plant or germplasm having resistance  Phakosora pachyrhizi . A soybean plant, part thereof and/or germplasm that has been identified, selected and/or produced by any of the methods of the present invention is also provided. Also provided are single nucleotide polymorphisms (SNPs) associated with resistance to pathogens; and compositions including amplification primer pairs capable of initiating DNA polymerization by a DNA polymerase on soybean nucleic acid templates to generate soybean marker amplicons.

RELATED APPLICATION INFORMATION

This application is a divisional application of U.S. application Ser.No. 16/405,365 filed May 7, 2019, which is a divisional application ofU.S. application Ser. No. 15/279,031 filed Sep. 28, 2016, now U.S. Pat.No. 10,306,853, issued on Jun. 4, 2019, which claims the benefit of U.S.Provisional Application No. 62/235,711, filed 1 Oct. 2015, the contentsof which are incorporated herein by reference.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled “80753 SEQ LISTING_ST25.txt”, 14.3 kilobytes in size,generated on Sep. 1, 2015 and filed via EFS-Web, is provided in lieu ofa paper copy. This Sequence Listing is hereby incorporated by referenceinto the specification for its disclosures.

FIELD OF THE INVENTION

The present invention relates to compositions and methods foridentifying, selecting and/or producing soybean plants having toleranceto Asian soy rust

BACKGROUND

Soybean (Glycine max L. Merr) is a major cash crop and investmentcommodity in North America and elsewhere. Soybean oil is one of the mostwidely used edible oils, and soybeans are used worldwide both in animalfeed and in human food production. Asian soy rust (ASR) in soybeans is awidespread problem in many parts of the world particularly in Brazil.

Different varieties of soybean vary in their sensitivity or tolerance toASR. Therefore, one of the most effective control measures is plantingAsian soy rust tolerant soybean varieties, and thus varietal selectionis important for the management of ASR. However, currently, determiningwhether a soybean cultivar might have tolerance to ASR typicallyinvolves testing each cultivar in the field or greenhouse underconditions that typically produce ASR symptoms. Thus, the presentinvention overcomes the shortcomings in the art by providing molecularmarkers associated with tolerance to ASR, thereby allowing thecharacterization of soybean cultivars for ASR by molecular analysisrather than phenotypic analysis.

SUMMARY OF THE INVENTION Description of the Figures

FIG. 1: shows rating scores for rust tolerance traits: (1) lesion typeand (2) rust severity

DEFINITIONS

Although the following terms are believed to be well understood by oneof ordinary skill in the art, the following definitions are set forth tofacilitate understanding of the presently disclosed subject matter.

As used herein, the terms “a” or “an” or “the” may refer to one or morethan one. For example, “a” marker (e.g., SNP, QTL, haplotype) can meanone marker or a plurality of markers (e.g., 2, 3, 4, 5, 6, and thelike).

As used herein, the term “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the term “about,” when used in reference to a measurablevalue such as an amount of mass, dose, time, temperature, and the like,is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1%of the specified amount.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention. Thus, the term “consisting essentially of” when used in aclaim of this invention is not intended to be interpreted to beequivalent to “comprising.”

As used herein, the term “allele” refers to one of two or more differentnucleotides or nucleotide sequences that occur at a specific locus.

A “locus” is a position on a chromosome where a gene or marker or alleleis located. In some embodiments, a locus may encompass one or morenucleotides.

As used herein, the terms “desired allele,” “target allele” and/or“allele of interest” are used interchangeably to refer to an alleleassociated with a desired trait. In some embodiments, a desired allelemay be associated with either an increase or a decrease (relative to acontrol) of or in a given trait, depending on the nature of the desiredphenotype.—In some embodiments of this invention, the phrase “desiredallele,” “target allele” or “allele of interest” refers to an allele(s)that is associated with tolerance to ASR in a soybean plant relative toa control soybean plant not having the target allele or alleles.

A marker is “associated with” a trait when said trait is linked to itand when the presence of the marker is an indicator of whether and/or towhat extent the desired trait or trait form will occur in aplant/germplasm comprising the marker. Similarly, a marker is“associated with” an allele or chromosome interval when it is linked toit and when the presence of the marker is an indicator of whether theallele or chromosome interval is present in a plant/germplasm comprisingthe marker. For example, “a marker associated with an ASR toleranceallele” refers to a marker whose presence or absence can be used topredict whether a plant will display tolerance to ASR.

As used herein, the term “ASR” or “Asian soy rust tolerance” refers to aplant's ability to endure and/or thrive despite being exposed to growthconditions in which ASR are low as compared to one or more controlplants (e.g., a plant lacking a marker associated with ASR).

Thus, “tolerance” in a soybean plant to ASR conditions is an indicationthat the soybean plant is less affected by the ASR conditions withrespect to yield, survivability and/or other relevant agronomicmeasures, compared to a less tolerant, more “susceptible” plant.Tolerance is a relative term, indicating that a “tolerant” soybean plantsurvives and/or produces a better yield in ASR growth conditions whencompared to a different (less tolerant) soybean plant (e.g., a differentsoybean strain or variety) grown in similar conditions ASR. That is,under ASR growth conditions a tolerant plant can have a greater survivalrate and/or yield, as compared to a soybean plant that is susceptible orintolerant to these ASR growth conditions. Asian soy rust “tolerance”sometimes can be used interchangeably with Asian soy rust “resistance”.Asian soy rust intolerant soybean varieties and cultivars are well knownin the art.

As used herein, the terms “backcross” and “backcrossing” refer to theprocess whereby a progeny plant is crossed back to one of its parentsone or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.). In abackcrossing scheme, the “donor” parent refers to the parental plantwith the desired gene or locus to be introgressed. The “recipient”parent (used one or more times) or “recurrent” parent (used two or moretimes) refers to the parental plant into which the gene or locus isbeing introgressed. For example, see Ragot, M. et al. Marker-assistedBackcrossing: A Practical Example, in TECHNIQUES ET UTILISATIONS DESMARQUEURS MOLECULAIRES LES COLLOQUES, Vol. 72, pp. 45-56 (1995); andOpenshaw et al., Marker-assisted Selection in Backcross Breeding, inPROCEEDINGS OF THE SYMPOSIUM “ANALYSIS OF MOLECULAR MARKER DATA,” pp.41-43 (1994). The initial cross gives rise to the F1 generation. Theterm “BC1” refers to the second use of the recurrent parent, “BC2”refers to the third use of the recurrent parent, and so on.

As used herein, the terms “cross” or “crossed” refer to the fusion ofgametes via pollination to produce progeny (e.g., cells, seeds orplants). The term encompasses both sexual crosses (the pollination ofone plant by another) and selfing (self-pollination, e.g., when thepollen and ovule are from the same plant). The term “crossing” refers tothe act of fusing gametes via pollination to produce progeny.

As used herein, the terms “cultivar” and “variety” refer to a group ofsimilar plants that by structural or genetic features and/or performancecan be distinguished from other varieties within the same species.

As used herein, the terms “elite” and/or “elite line” refer to any linethat is substantially homozygous and has resulted from breeding andselection for desirable agronomic performance.

As used herein, the terms “exotic,” “exotic line” and “exotic germplasm”refer to any plant, line or germplasm that is not elite. In general,exotic plants/germplasms are not derived from any known elite plant orgermplasm, but rather are selected to introduce one or more desiredgenetic elements into a breeding program (e.g., to introduce novelalleles into a breeding program).

A “genetic map” is a description of genetic linkage relationships amongloci on one or more chromosomes within a given species, generallydepicted in a diagrammatic or tabular form. For each genetic map,distances between loci are measured by the recombination frequenciesbetween them. Recombination between loci can be detected using a varietyof markers. A genetic map is a product of the mapping population, typesof markers used, and the polymorphic potential of each marker betweendifferent populations. The order and genetic distances between loci candiffer from one genetic map to another.

As used herein, the term “genotype” refers to the genetic constitutionof an individual (or group of individuals) at one or more genetic loci,as contrasted with the observable and/or detectable and/or manifestedtrait (the phenotype). Genotype is defined by the allele(s) of one ormore known loci that the individual has inherited from its parents. Theterm genotype can be used to refer to an individual's geneticconstitution at a single locus, at multiple loci, or more generally, theterm genotype can be used to refer to an individual's genetic make-upfor all the genes in its genome. Genotypes can be indirectlycharacterized, e.g., using markers and/or directly characterized bynucleic acid sequencing.

As used herein, the term “germplasm” refers to genetic material of orfrom an individual (e.g., a plant), a group of individuals (e.g., aplant line, variety or family), or a clone derived from a line, variety,species, or culture. The germplasm can be part of an organism or cell,or can be separate from the organism or cell. In general, germplasmprovides genetic material with a specific genetic makeup that provides afoundation for some or all of the hereditary qualities of an organism orcell culture. As used herein, germplasm includes cells, seed or tissuesfrom which new plants may be grown, as well as plant parts that can becultured into a whole plant (e.g., leaves, stems, buds, roots, pollen,cells, etc.).

A “haplotype” is the genotype of an individual at a plurality of geneticloci, i.e., a combination of alleles. Typically, the genetic loci thatdefine a haplotype are physically and genetically linked, i.e., on thesame chromosome segment. The term “haplotype” can refer to polymorphismsat a particular locus, such as a single marker locus, or polymorphismsat multiple loci along a chromosomal segment.

As used herein, the term “heterozygous” refers to a genetic statuswherein different alleles reside at corresponding loci on homologouschromosomes.

As used herein, the term “homozygous” refers to a genetic status whereinidentical alleles reside at corresponding loci on homologouschromosomes.

As used herein, the term “hybrid” in the context of plant breedingrefers to a plant that is the offspring of genetically dissimilarparents produced by crossing plants of different lines or breeds orspecies, including but not limited to the cross between two inbredlines.

As used herein, the term “inbred” refers to a substantially homozygousplant or variety. The term may refer to a plant or plant variety that issubstantially homozygous throughout the entire genome or that issubstantially homozygous with respect to a portion of the genome that isof particular interest.

As used herein, the term “indel” refers to an insertion or deletion in apair of nucleotide sequences, wherein a first sequence may be referredto as having an insertion relative to a second sequence or the secondsequence may be referred to as having a deletion relative to the firstsequence.

As used herein, the terms “introgression,” “introgressing” and“introgressed” refer to both the natural and artificial transmission ofa desired allele or combination of desired alleles of a genetic locus orgenetic loci from one genetic background to another. For example, adesired allele at a specified locus can be transmitted to at least oneprogeny via a sexual cross between two parents of the same species,where at least one of the parents has the desired allele in its genome.Alternatively, for example, transmission of an allele can occur byrecombination between two donor genomes, e.g., in a fused protoplast,where at least one of the donor protoplasts has the desired allele inits genome. The desired allele may be a selected allele of a marker, aQTL, a transgene, or the like. Offspring comprising the desired allelecan be backcrossed one or more times (e.g., 1, 2, 3, 4, or more times)to a line having a desired genetic background, selecting for the desiredallele, with the result being that the desired allele becomes fixed inthe desired genetic background. For example, a marker associated withASR tolerance may be introgressed from a donor into a recurrent parentthat is ASR. The resulting offspring could then be backcrossed one ormore times and selected until the progeny possess the genetic marker(s)associated with ASR in the recurrent parent background.

As used herein, the term “linkage” refers to the degree with which onemarker locus is associated with another marker locus or some other. Thelinkage relationship between a genetic marker and a phenotype may begiven as a “probability” or “adjusted probability.” Linkage can beexpressed as a desired limit or range. For example, in some embodiments,any marker is linked (genetically and physically) to any other markerwhen the markers are separated by less than about 50, 40, 30, 25, 20, or15 map units (or cM).

A centimorgan (“cM”) or a genetic map unit (m.u.) is a unit of measureof recombination frequency and is defined as the distance between genesfor which one product of meiosis in 100 is recombinant. One cM is equalto a 1% chance that a marker at one genetic locus will be separated froma marker at a second locus due to crossing over in a single generation.Thus, a recombinant frequency (RF) of 1% is equivalent to 1 m.u.

As used herein, the phrase “linkage group” refers to all of the genes orgenetic traits that are located on the same chromosome. Within thelinkage group, those loci that are close enough together can exhibitlinkage in genetic crosses. Since the probability of crossover increaseswith the physical distance between loci on a chromosome, loci for whichthe locations are far removed from each other within a linkage groupmight not exhibit any detectable linkage in direct genetic tests. Theterm “linkage group” is mostly used to refer to genetic loci thatexhibit linked behavior in genetic systems where chromosomal assignmentshave not yet been made. Thus, the term “linkage group” is synonymouswith the physical entity of a chromosome, although one of ordinary skillin the art will understand that a linkage group can also be defined ascorresponding to a region of (i.e., less than the entirety) of a givenchromosome.

As used herein, the term “linkage disequilibrium” refers to a non-randomsegregation of genetic loci or traits (or both). In either case, linkagedisequilibrium implies that the relevant loci are within sufficientphysical proximity along a length of a chromosome so that they segregatetogether with greater than random (i.e., non-random) frequency (in thecase of co-segregating traits, the loci that underlie the traits are insufficient proximity to each other). Markers that show linkagedisequilibrium are considered linked. Linked loci co-segregate more than50% of the time, e.g., from about 51% to about 100% of the time. Inother words, two markers that co-segregate have a recombinationfrequency of less than 50% (and, by definition, are separated by lessthan 50 cM on the same chromosome). As used herein, linkage can bebetween two markers, or alternatively between a marker and a phenotype.A marker locus can be “associated with” (linked to) a trait, e.g., ASR.The degree of linkage of a genetic marker to a phenotypic trait ismeasured, e.g., as a statistical probability of co-segregation of thatmarker with the phenotype.

Linkage disequilibrium is most commonly assessed using the measure r²,which is calculated using the formula described by Hill and Robertson,Theor. Appl. Genet. 38:226 (1968). When r²=1, complete linkagedisequilibrium exists between the two marker loci, meaning that themarkers have not been separated by recombination and have the sameallele frequency. Values for r² above ⅓ indicate sufficiently stronglinkage disequilibrium to be useful for mapping. Ardlie et al., NatureReviews Genetics 3:299 (2002). Hence, alleles are in linkagedisequilibrium when r² values between pairwise marker loci are greaterthan or equal to about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

As used herein, the term “linkage equilibrium” describes a situationwhere two markers independently segregate, i.e., sort among progenyrandomly. Markers that show linkage equilibrium are considered unlinked(whether or not they lie on the same chromosome).

As used herein, the terms “marker” and “genetic marker” are usedinterchangeably to refer to a nucleotide and/or a nucleotide sequencethat has been associated with a phenotype and/or trait. A marker may be,but is not limited to, an allele, a gene, a haplotype, a chromosomeinterval, a restriction fragment length polymorphism (RFLP), a simplesequence repeat (SSR), a random amplified polymorphic DNA (RAPD), acleaved amplified polymorphic sequence (CAPS) (Rafalski and Tingey,Trends in Genetics 9:275 (1993)), an amplified fragment lengthpolymorphism (AFLP) (Vos et al., Nucleic Acids Res. 23:4407 (1995)), asingle nucleotide polymorphism (SNP) (Brookes, Gene 234:177 (1993)), asequence-characterized amplified region (SCAR) (Paran and Michelmore,Theor. Appl. Genet. 85:985 (1993)), a sequence-tagged site (STS)(Onozaki et al., Euphytica 138:255 (2004)), a single-strandedconformation polymorphism (SSCP) (Orita et al., Proc. Natl. Acad. Sci.USA 86:2766 (1989)), an inter-simple sequence repeat (ISSR) (Blair etal., Theor. Appl. Genet. 98:780 (1999)), an inter-retrotransposonamplified polymorphism (IRAP), a retrotransposon-microsatelliteamplified polymorphism (REMAP) (Kalendar et al., Theor. Appl. Genet.98:704 (1999)), an isozyme marker, an RNA cleavage product (such as aLynx tag) or any combination of the markers described herein. A markermay be present in genomic or expressed nucleic acids (e.g., ESTs). Alarge number of soybean genetic markers are known in the art, and arepublished or available from various sources, such as the SoyBaseinternet resource (www.soybase.org). In some embodiments, a geneticmarker of this invention is an SNP allele, a SNP allele located in achromosome interval and/or a haplotype (combination of SNP alleles) eachof which is associated with Asian soy rust tolerance.

Markers corresponding to genetic polymorphisms between members of apopulation can be detected by methods well-established in the art. Theseinclude, but are not limited to, nucleic acid sequencing, hybridizationmethods, amplification methods (e.g., PCR-based sequence specificamplification methods), detection of restriction fragment lengthpolymorphisms (RFLP), detection of isozyme markers, detection ofpolynucleotide polymorphisms by allele specific hybridization (ASH),detection of amplified variable sequences of the plant genome, detectionof self-sustained sequence replication, detection of simple sequencerepeats (SSRs), detection of randomly amplified polymorphic DNA (RAPD),detection of single nucleotide polymorphisms (SNPs), and/or detection ofamplified fragment length polymorphisms (AFLPs). Thus, in someembodiments of this invention, such well known methods can be used todetect the SNP alleles as defined herein (See, e.g., Tables 1-3)

Accordingly, in some embodiments of this invention, a marker is detectedby amplifying a Glycine sp. nucleic acid with two oligonucleotideprimers by, for example, the polymerase chain reaction (PCR).

A “marker allele,” also described as an “allele of a marker locus,” canrefer to one of a plurality of polymorphic nucleotide sequences found ata marker locus in a population that is polymorphic for the marker locus.

“Marker-assisted selection” (MAS) is a process by which phenotypes areselected based on marker genotypes. Marker assisted selection includesthe use of marker genotypes for identifying plants for inclusion inand/or removal from a breeding program or planting.

As used herein, the terms “marker locus” and “marker loci” refer to aspecific chromosome location or locations in the genome of an organismwhere a specific marker or markers can be found. A marker locus can beused to track the presence of a second linked locus, e.g., a linkedlocus that encodes or contributes to expression of a phenotypic trait.For example, a marker locus can be used to monitor segregation ofalleles at a locus, such as a QTL or single gene, that are geneticallyor physically linked to the marker locus.

As used herein, the terms “marker probe” and “probe” refer to anucleotide sequence or nucleic acid molecule that can be used to detectthe presence of one or more particular alleles within a marker locus(e.g., a nucleic acid probe that is complementary to all of or a portionof the marker or marker locus, through nucleic acid hybridization).Marker probes comprising about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100 or more contiguous nucleotides may be used for nucleic acidhybridization. Alternatively, in some aspects, a marker probe refers toa probe of any type that is able to distinguish (i.e., genotype) theparticular allele that is present at a marker locus. Non-limitingexamples of probes of this invention include SEQ ID NOs:19-54 and137-300.

As used herein, the term “molecular marker” may be used to refer to agenetic marker, as defined above, or an encoded product thereof (e.g., aprotein) used as a point of reference when identifying a linked locus. Amolecular marker can be derived from genomic nucleotide sequences orfrom expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA,etc.). The term also refers to nucleotide sequences complementary to orflanking the marker sequences, such as nucleotide sequences used asprobes and/or primers capable of amplifying the marker sequence.Nucleotide sequences are “complementary” when they specificallyhybridize in solution, e.g., according to Watson-Crick base pairingrules. Some of the markers described herein can also be referred to ashybridization markers when located on an indel region. This is becausethe insertion region is, by definition, a polymorphism vis-ã-vis a plantwithout the insertion. Thus, the marker need only indicate whether theindel region is present or absent. Any suitable marker detectiontechnology may be used to identify such a hybridization marker, e.g.,SNP technology.

As used herein, the term “primer” refers to an oligonucleotide which iscapable of annealing to a nucleic acid target and serving as a point ofinitiation of DNA synthesis when placed under conditions in whichsynthesis of a primer extension product is induced (e.g., in thepresence of nucleotides and an agent for polymerization such as DNApolymerase and at a suitable temperature and pH). A primer (in someembodiments an extension primer and in some embodiments an amplificationprimer) is in some embodiments single stranded for maximum efficiency inextension and/or amplification. In some embodiments, the primer is anoligodeoxyribonucleotide. A primer is typically sufficiently long toprime the synthesis of extension and/or amplification products in thepresence of the agent for polymerization. The minimum lengths of theprimers can depend on many factors, including, but not limited totemperature and composition (A/T vs. G/C content) of the primer. In thecontext of amplification primers, these are typically provided as a pairof bi-directional primers consisting of one forward and one reverseprimer or provided as a pair of forward primers as commonly used in theart of DNA amplification such as in PCR amplification. As such, it willbe understood that the term “primer”, as used herein, can refer to morethan one primer, particularly in the case where there is some ambiguityin the information regarding the terminal sequence(s) of the targetregion to be amplified. Hence, a “primer” can include a collection ofprimer oligonucleotides containing sequences representing the possiblevariations in the sequence or includes nucleotides which allow a typicalbase pairing.

Primers can be prepared by any suitable method. Methods for preparingoligonucleotides of specific sequence are known in the art, and include,for example, cloning and restriction of appropriate sequences and directchemical synthesis. Chemical synthesis methods can include, for example,the phospho di- or tri-ester method, the diethylphosphoramidate methodand the solid support method disclosed in U.S. Pat. No. 4,458,066.

Primers can be labeled, if desired, by incorporating detectable moietiesby for instance spectroscopic, fluorescence, photochemical, biochemical,immunochemical, or chemical moieties.

The PCR method is well described in handbooks and known to the skilledperson. After amplification by PCR, target polynucleotides can bedetected by hybridization with a probe polynucleotide which forms astable hybrid with that of the target sequence under stringent tomoderately stringent hybridization and wash conditions. If it isexpected that the probes are essentially completely complementary (i.e.,about 99% or greater) to the target sequence, stringent conditions canbe used. If some mismatching is expected, for example if variant strainsare expected with the result that the probe will not be completelycomplementary, the stringency of hybridization can be reduced. In someembodiments, conditions are chosen to rule out non-specific/adventitiousbinding. Conditions that affect hybridization, and that select againstnon-specific binding are known in the art, and are described in, forexample, Sambrook & Russell (2001). Molecular Cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., United States of America. Generally, lower saltconcentration and higher temperature hybridization and/or washesincrease the stringency of hybridization conditions.

As used herein, the term “probe” refers to a single-strandedoligonucleotide sequence that will form a hydrogen-bonded duplex with acomplementary sequence in a target nucleic acid sequence analyte or itscDNA derivative.

Different nucleotide sequences or polypeptide sequences having homologyare referred to herein as “homologues.” The term homologue includeshomologous sequences from the same and other species and orthologoussequences from the same and other species. “Homology” refers to thelevel of similarity between two or more nucleotide sequences and/oramino acid sequences in terms of percent of positional identity (i.e.,sequence similarity or identity). Homology also refers to the concept ofsimilar functional properties among different nucleic acids, aminoacids, and/or proteins.

As used herein, the phrase “nucleotide sequence homology” refers to thepresence of homology between two polynucleotides. Polynucleotides have“homologous” sequences if the sequence of nucleotides in the twosequences is the same when aligned for maximum correspondence. The“percentage of sequence homology” for polynucleotides, such as 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100 percent sequencehomology, can be determined by comparing two optimally aligned sequencesover a comparison window (e.g., about 20-200 contiguous nucleotides),wherein the portion of the polynucleotide sequence in the comparisonwindow can include additions or deletions (i.e., gaps) as compared to areference sequence for optimal alignment of the two sequences. Optimalalignment of sequences for comparison can be conducted by computerizedimplementations of known algorithms, or by visual inspection. Readilyavailable sequence comparison and multiple sequence alignment algorithmsare, respectively, the Basic Local Alignment Search Tool (BLAST;Altschul et al. (1990) J Mol Biol 215:403-10; Altschul et al. (1997)Nucleic Acids Res 25:3389-3402) and ClustalX (Chenna et al. (2003)Nucleic Acids Res 31:3497-3500) programs, both available on theInternet. Other suitable programs include, but are not limited to, GAP,BestFit, PlotSimilarity, and FASTA, which are part of the Accelrys GCGPackage available from Accelrys Software, Inc. of San Diego, Calif.,United States of America.

As used herein “sequence identity” refers to the extent to which twooptimally aligned polynucleotide or polypeptide sequences are invariantthroughout a window of alignment of components, e.g., nucleotides oramino acids. “Identity” can be readily calculated by known methodsincluding, but not limited to, those described in: ComputationalMolecular Biology (Lesk, A. M., ed.) Oxford University Press, New York(1988); Biocomputing: Informatics and Genome Projects (Smith, D. W.,ed.) Academic Press, New York (1993); Computer Analysis of SequenceData, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press,New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje,G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov,M. and Devereux, J., eds.) Stockton Press, New York (1991).

As used herein, the term “substantially identical” or “corresponding to”means that two nucleotide sequences have at least 50%, 60%, 70%, 75%,80%, 85%, 90% or 95% sequence identity. In some embodiments, the twonucleotide sequences can have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity.

An “identity fraction” for aligned segments of a test sequence and areference sequence is the number of identical components which areshared by the two aligned sequences divided by the total number ofcomponents in the reference sequence segment, i.e., the entire referencesequence or a smaller defined part of the reference sequence. Percentsequence identity is represented as the identity fraction multiplied by100. As used herein, the term “percent sequence identity” or “percentidentity” refers to the percentage of identical nucleotides in a linearpolynucleotide sequence of a reference (“query”) polynucleotide molecule(or its complementary strand) as compared to a test (“subject”)polynucleotide molecule (or its complementary strand) when the twosequences are optimally aligned (with appropriate nucleotide insertions,deletions, or gaps totaling less than 20 percent of the referencesequence over the window of comparison). In some embodiments, “percentidentity” can refer to the percentage of identical amino acids in anamino acid sequence.

Optimal alignment of sequences for aligning a comparison window is wellknown to those skilled in the art and may be conducted by tools such asthe local homology algorithm of Smith and Waterman, the homologyalignment algorithm of Needleman and Wunsch, the search for similaritymethod of Pearson and Lipman, and optionally by computerizedimplementations of these algorithms such as GAP, BESTFIT, FASTA, andTFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc.,Burlington, Mass.). The comparison of one or more polynucleotidesequences may be to a full-length polynucleotide sequence or a portionthereof, or to a longer polynucleotide sequence. For purposes of thisinvention “percent identity” may also be determined using BLASTX version2.0 for translated nucleotide sequences and BLASTN version 2.0 forpolynucleotide sequences.

The percent of sequence identity can be determined using the “Best Fit”or “Gap” program of the Sequence Analysis Software Package™ (Version 10;Genetics Computer Group, Inc., Madison, Wis.). “Gap” utilizes thealgorithm of Needleman and Wunsch (Needleman and Wunsch, J Mol. Biol.48:443-453, 1970) to find the alignment of two sequences that maximizesthe number of matches and minimizes the number of gaps. “BestFit”performs an optimal alignment of the best segment of similarity betweentwo sequences and inserts gaps to maximize the number of matches usingthe local homology algorithm of Smith and Waterman (Smith and Waterman,Adv. Appl. Math., 2:482-489, 1981, Smith et al., Nucleic Acids Res.11:2205-2220, 1983).

Useful methods for determining sequence identity are also disclosed inGuide to Huge Computers (Martin J. Bishop, ed., Academic Press, SanDiego (1994)), and Carillo et al. (Applied Math 48:1073(1988)). Moreparticularly, preferred computer programs for determining sequenceidentity include but are not limited to the Basic Local Alignment SearchTool (BLAST) programs which are publicly available from National CenterBiotechnology Information (NCBI) at the National Library of Medicine,National Institute of Health, Bethesda, Md. 20894; see BLAST Manual,Altschul et al., NCBI, NLM, NIH; (Altschul et al., J. Mol. Biol.215:403-410 (1990)); version 2.0 or higher of BLAST programs allows theintroduction of gaps (deletions and insertions) into alignments; forpeptide sequence BLASTX can be used to determine sequence identity; andfor polynucleotide sequence BLASTN can be used to determine sequenceidentity.

As used herein, the terms “phenotype,” “phenotypic trait” or “trait”refer to one or more traits of an organism. The phenotype can beobservable to the naked eye, or by any other means of evaluation knownin the art, e.g., microscopy, biochemical analysis, or anelectromechanical assay. In some cases, a phenotype is directlycontrolled by a single gene or genetic locus, i.e., a “single genetrait.” In other cases, a phenotype is the result of several genes.

As used herein, the term “polymorphism” refers to a variation in thenucleotide sequence at a locus, where said variation is too common to bedue merely to a spontaneous mutation. A polymorphism must have afrequency of at least about 1% in a population. A polymorphism can be asingle nucleotide polymorphism (SNP), or an insertion/deletionpolymorphism, also referred to herein as an “indel.” Additionally, thevariation can be in a transcriptional profile or a methylation pattern.The polymorphic site or sites of a nucleotide sequence can be determinedby comparing the nucleotide sequences at one or more loci in two or moregermplasm entries.

As used herein, the term “plant” can refer to a whole plant, any partthereof, or a cell or tissue culture derived from a plant. Thus, theterm “plant” can refer to a whole plant, a plant component or a plantorgan (e.g., leaves, stems, roots, etc.), a plant tissue, a seed and/ora plant cell. A plant cell is a cell of a plant, taken from a plant, orderived through culture from a cell taken from a plant.

As used herein, the term “soybean” refers to a plant, and any partthereof, of the genus Glycine including, but not limited to Glycine max.

As used herein, the term “plant part” includes but is not limited toembryos, pollen, seeds, leaves, flowers (including but not limited toanthers, ovules and the like), fruit, stems or branches, roots, roottips, cells including cells that are intact in plants and/or parts ofplants, protoplasts, plant cell tissue cultures, plant calli, plantclumps, and the like. Thus, a plant part includes soybean tissue culturefrom which soybean plants can be regenerated. Further, as used herein,“plant cell” refers to a structural and physiological unit of the plant,which comprises a cell wall and also may refer to a protoplast. A plantcell of the present invention can be in the form of an isolated singlecell or can be a cultured cell or can be a part of a higher-organizedunit such as, for example, a plant tissue or a plant organ.

As used herein, the term “population” refers to a geneticallyheterogeneous collection of plants sharing a common genetic derivation.

As used herein, the terms “progeny”, “progeny plant,” and/or “offspring”refer to a plant generated from a vegetative or sexual reproduction fromone or more parent plants. A progeny plant may be obtained by cloning orselfing a single parent plant, or by crossing two parental plants andincludes selfings as well as the F1 or F2 or still further generations.An F1 is a first-generation offspring produced from parents at least oneof which is used for the first time as donor of a trait, while offspringof second generation (F2) or subsequent generations (F3, F4, and thelike) are specimens produced from selfings or crossings of F1s, F2s andthe like. An F1 can thus be (and in some embodiments is) a hybridresulting from a cross between two true breeding parents (the phrase“true-breeding” refers to an individual that is homozygous for one ormore traits), while an F2 can be (and in some embodiments is) anoffspring resulting from self-pollination of the F1 hybrids.

As used herein, the term “reference sequence” refers to a definednucleotide sequence used as a basis for nucleotide sequence comparison.The reference sequence for a marker, for example, can be obtained bygenotyping a number of lines at the locus or loci of interest, aligningthe nucleotide sequences in a sequence alignment program, and thenobtaining the consensus sequence of the alignment. Hence, a referencesequence identifies the polymorphisms in alleles at a locus. A referencesequence may not be a copy of an actual nucleic acid sequence from anyparticular organism; however, it is useful for designing primers andprobes for actual polymorphisms in the locus or loci.

Genetic Mapping

Genetic loci correlating with particular phenotypes, such as ASR, can bemapped in an organism's genome. By identifying a marker or cluster ofmarkers that co-segregate with a trait of interest, the breeder is ableto rapidly select a desired phenotype by selecting for the proper marker(a process called marker-assisted selection, or MAS). Such markers mayalso be used by breeders to design genotypes in silico and to practicewhole genome selection.

The present invention provides markers associated with ASR in soybean.

Detection of these markers and/or other linked markers can be used toidentify, select and/or produce soybean plants having ASR and/or toeliminate soybean plants from breeding programs or from planting that donot have ASR.

Markers Associated with ASR

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization can be due to DNA-DNAhybridization techniques after digestion with a restriction enzyme(e.g., an RFLP) and/or due to techniques using the polymerase chainreaction (e.g., SNP, STS, SSR/microsatellites, AFLP, and the like). Insome embodiments, all differences between two parental genotypessegregate in a mapping population based on the cross of these parentalgenotypes. The segregation of the different markers can be compared andrecombination frequencies can be calculated. Methods for mapping markersin plants are disclosed in, for example, Glick & Thompson (1993) Methodsin Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton,Fla., United States of America; Zietkiewicz et al. (1994) Genomics20:176-183.

In one embodiment of the invention, it is contemplated that one may usegene editing technologies (e.g. Talen, Meganucleases, CRISPR, etc.) tointroduce a favorable allele introduction for ASR resistance into asoybean germplasm not comprising said favorable allele wherein thefavorable allele is on chromosome 18 and within 20 cM, 10 cM, 5 cM orless from a chromosomal interval comprising physical positions58,722,971-60,910,083 or any favorable allele or favorable haplotype asdescribed in Tables 1 or 2.

Table 1 provides information about the ASR associated markers presentedincluding the physical location of the marker on the respective soybeanchromosome, and the target allele that is associated with ASR. Table 2gives the names of the associated markers (SNPs) of this invention, thespecific associated ASR trait or traits, the physical genetic locationsof each marker on the respective soybean chromosome or linkage group,and the target allele that is associated with ASR.

Markers of the present invention are described herein with respect tothe positions of marker loci in the 8X public build of the Williams82soybean genome at the SoyBase internet resource(www.soybase.org/SequenceIntro.php) or USDA at(bfgl.anri.barc.usda.gov/cgi-bin/soybean/Linkage.pl). See Table 1 below.

TABLE 1 The respective soybean chromosome or linkage group of physicaland genetic positions including the sequence identifiers for the DNAfragments comprising the SNPs or indels and two probes sequences withtagged SNP allele for each assay for the genetic markers presented.Physical SEQ ID Favorable allele Probe 1 Probe 2 Linkage position NO for(respective SNP SEQ ID SEQ ID Group SNP in fragment position in NO NOPrimer Assay (chromosome Willams 82 comprising fragment of (detected(detected SEQ ID Name #) genome marker column 5) nucleotide) nucleotide)NO's SY0707 G (18) 604693 1 A (428)  8 (G)  9 (A) 22, 23 A 65 SY0708 G(18) 604881 2 A (806) 10 (A) 11 (G) 24, 25 A 41 SY0714 G (18) 605777 3 A(895) 12 (A) 13 (G) 26, 27 DQ 61 SY3765 G (18) 603204 4 G (300) 14 (G)15 (A) 28, 29 84 SY3771 G (18) 606348 5 T (301) 16 (A) 17 (T) 30, 31 61SY3776 G (18) 607803 6 A (300) 18 (G) 19 (A) 32, 33 64 SY3777 G (18)608026 7 A (301) 20 (A) 21 (G) 34, 35 07

TABLE 2 Shows respective plant introduction lines (rust tolerant andsusceptible) and respective haplotypes for each. The Table further showslesion types (TAN or RB1, see FIG. 1) SNP assay Lesion SY3765 SY0707ASY0708A SY0714DQ SY3771 SY3776 SY3777 type in Locus/ Physical Mapposition PI # Brazil haplotype 60.320.484 60.469.365 60.488.14160.577.761 60.634.861 60.780.364 60.802.607 PI 200492 TAN Rpp1-hap1 G AA A T A G PI 368038 TAN Rpp1-hap1 G A A A T A G PI 368039 TAN Rpp1-hap1G A A A T A G PI 547875 TAN Rpp1-hap1 G A A A T A G PI 594754 RB1Rpp1-hap2 A G A A T G G PI 594760 RB1 Rpp1-hap2 A G A A T G G B PI594767 RB1 Rpp1-hap2 A G A A T G G A PI 587880 RB1 Rpp1b G A A A A G A API 587905 RB1 Rpp1b G A A A A G A PI 561356 RB1 Rpp1b G A A A A G API594538A RB1 Rpp1b G A A A A G A BA821791 TAN susceptible A G G G T A GBENNING TAN susceptible A G G G A A G

The above examples clearly illustrate the advantages of the invention.Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

Throughout this application, various patents, patent publications andnon-patent publications are referenced. The disclosures of thesepatents, patent publications and non-patent publications in theirentireties are incorporated by reference herein into this application inorder to more fully describe the state of the art to which thisinvention pertains.

That which is claimed:
 1. A method of identifying an Asian Soy Rust(ASR) tolerant soybean plant or part thereof, comprising the steps of:a. isolating a nucleic acid from a soybean plant or part thereof; b.detecting in said nucleic acid, the presence of a marker associated withASR tolerance in a soybean plant, wherein said marker is located withina chromosomal interval on soybean chromosome 18 corresponding tophysical positions 58,722,971-60,910,083; and c. thereby identifying aASR tolerant soybean plant or part thereof.
 2. The method of claim 1,wherein the chromosomal interval comprises an allele associated with ASRtolerance wherein said allele corresponds to both of an A at position428 of SEQ ID NO: 1 and an A at position 806 of SEQ ID NO:
 2. 3. Themethod of claim 2, wherein the chromosomal interval further comprises anallele associated with ASR tolerance wherein said allele corresponds toany of an A at position 895 of SEQ ID NO: 3; a G at position 300 of SEQID NO: 4; a T at position 301 of SEQ ID NO: 5; an A at position 300 ofSEQ ID NO: 6; an A at position 301 of SEQ ID NO: 7; or any combinationthereof.
 4. The method of claim 1, wherein the soybean plant is an elitesoybean plant.
 5. The method of claim 1, wherein the chromosomalinterval is derived from any of PI200492, PI368038, PI368039, PI547875,PI594754, PI594760B, PI594767A, PI587880A, PI587905, PI561356,PI594538A, and a progeny thereof.
 6. An ASR tolerant soybean plantidentified by the method of claim
 1. 7. An ASR tolerant progeny plant ofthe ASR tolerant soybean plant of claim 6, the progeny plant having themarker associated with ASR tolerance introduced into its genome from theASR tolerant soybean plant via a breeding program.